© 2019 by the Serbian Biological Society 159
The influence of rs53576 and rs2254298 oxytocin receptor gene polymorphisms on
plasma oxytocin levels and measures of empathy
Jelena Bašić1,*, Vuk Milošević2, Miloš Stanković3,4, Tatjana Jevtović-Stoimenov1, Tatjana Cvetković1,
Milena Despotović1 and Dušica Pavlović1
1University of Niš, Faculty of Medicine, Department of Biochemistry, Serbia
2Clinic of Neurology, Clinical Center Niš, Serbia
3Department of Cognitive and Clinical Neuroscience, Central Institute of Mental Health, Medical Faculty Mannheim,
Heidelberg University, Mannheim, Germany
4University of Niš, Faculty of Philosophy, Serbia
*Corresponding author: [email protected]
Received: December 6, 2018; Revised: December 13, 2018; Accepted: December 14, 2018; Published online: December 20, 2018
Abstract: Oxytocin achieves its effects after binding the oxytocin receptor (OXTR). Oxytocin plays an important role in
empathy. The aim of this study was to examine the influence of two single nucleotide polymorphisms (SNPs) of the OXTR
gene (rs53576 and rs2254298) on empathy measures and plasma oxytocin levels. Seventy-four university students were
screened for the OXTR rs53576 and rs2254298 SNPs using the polymerase chain reaction-restriction fragment length
poly-morphism (PCR-RFLP) method. The level of oxytocin in the plasma was measured by the enzyme linked immunosorbent
assay (ELISA). All subjects were assessed with the empathy quotient(EQ) questionnaire and Reading the Mind in the Eyes
Test (RMET). Subjects carrying the rs53576 GG genotype had a higher EQ score, compared to GA/AA genotype carriers. By gender analysis, significance was reached only in females. Considering the influence of both examined polymorphisms on empathy, participants with the GGGG diplotype had a higher EQ in comparison with GAGG/AAGG carriers. These hormone effects were not correlated with plasma oxytocin levels. This is the first study implying that higher empathy in rs53576 GG genotype carriers may not depend on plasma oxytocin levels, but possibly on the number and function of OXTRs in the brain.
Keywords: empathy; oxytocin; oxytocin receptor; rs53576 SNP; rs2254298 SNP
How to cite this article: Bašić J, Milošević V, Stanković M, Jevtović-Stoimenov T, Cvetković T, Despotović M, Pavlović D. The influence of rs53576 and rs2254298 oxytocin receptor gene polymorphisms on plasma oxytocin levels and measures of empathy. Arch Biol Sci. 2019;71(1):159-65.
INTRODUCTION
Oxytocin is a nonapeptide produced as a prohormone in the supraoptic and paraventricular nuclei of the hypothalamus, from where it is transported by axonal transport to the posterior pituitary gland. The inactive precursor is hydrolyzed into smaller fragments, one of which is neurophysin I. Peptidyl glycine α-amidating monooxygenase (PAM) enables the release of active oxytocin into the circulation [1]. Oxytocin achieves its effects after binding to the oxytocin receptor (OXTR), a polypeptide consisting of 389 amino acids that be-longs to the class I G-protein-coupled receptor fam-ily. The OXTR is synthesized in the uterus, mam-mary gland, placenta, ovaries, testes, heart muscle and kidneys. In the brain, this receptor is expressed
basis of empathy and biomarkers that could play an important role in this process is the subject of interest of an increasing number of authors. There are some data that genetic variations in the OXTR gene could influence receptor function, the binding of oxytocin and the effects of the hormone. The OXTR geneis found on chromosome 3p25 and it consists of 4 exons and 3 introns [5]. The two most commonly studied single nucleotide polymorphisms (SNPs) are rs53576 (6930G>A) and rs2254298 (9073G>A) located in in-tron 3 of the OXTR gene. Previous studies indicate the association of both SNPs with autism [6,7], unipolar depression and separation anxiety [8]. Studies that examined the effect of the OXTR rs53576 SNP on empathy provided contradictory results [9,10]. Feld-man et al. [11] showed that the OXTR rs2254298 risk allele was associated with lower plasma oxytocin levels and was related to less parental touch, indicating that future studies should include the rs53576 SNP and its effects. However, to the authors’ knowledge, no data is available on how the diplotypes of both SNPs (OXTR
rs53576 and rs2254298) affect empathy, or whether the influence of both SNPs on empathy depends on plasma oxytocin levels. The aim of this study was to examine the distribution of the rs53576 and rs2254298 genetic variations in the OXTR gene in university students, and to examine the impact of these SNPs on empathy measures and plasma oxytocin levels.
MATERIALS AND METHODS
Ethics statement
All participants voluntarily agreed and signed an in-formed consent to participate in the research. The Ethical Committee of the Medical Faculty, University of Niš, provided consent for conducting this study. There was no compensation for participation in the study, and the participants were treated in accordance with the Helsinki Declaration.
Participants
The study involved 74 students at the Medical Faculty, University of Niš, Serbia. The participants included 32 men (Mage=21.06, SD=1.26) and 42 women (Mage=21.05, SD=1.51), without self-reported histories of chronic
disease, psychiatric disorders, drug addiction, preg-nancy (for females), and none were parents. The re-search was conducted in the Laboratory for Functional Genomics and Proteomics at the Medical Faculty, University of Niš, Serbia.
Blood sample preparation and genotyping
Blood samples were taken between 9:00 and 10:00 am. From all blood samples (with EDTA as the anticoagu-lant), 200 μL of blood were separated and used for DNA isolation. The blood samples were then centrifuged at 2000 x g for 10 min at +4°C, after which the plasma was separated and frozen at -80°C.
The isolation of DNA was performed using a commercial kit for DNA isolation (QIAamp DNA Blood Mini Kit, Qiagen GmbH, Hilden, Germany). We examined the following SNPs in the OXTR gene: rs53576 (6930G>A) and rs2254298 (9073G>A), by the PCR-RFLP method. The fragments (340 base pairs (bp) (rs53576) and 307 bp (rs2254298)) were amplified using forward and reverse primers (F: 5΄-GCCCAC-CATGCTCTCCACATC-3΄ and R: 5΄- GCTGGACT-CAGGAGGAATAGGGAC-3’ for the rs53576 SNP and F: 5΄-TGAAAGCAGAGGTTGTGTGGACAGG-3’ and R: 5’-AACGCCCACCCCAGTTTCTTC-3’ for the rs2254298 SNP). The PCR reaction mixture in a volume of 25 μL contained: 12.5 μL of KAPA 2G Fast HS Ready-Mix PCR kit solution (KAPA Biosystems, Germany), 0.5 μL of primer (10 pmol/μL) (Fermentas GmbH, St. Leon-Rot, Germany) and 20 ng of DNA. The PCR conditions were as follows: initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, elongation at 72°C for 15 s and termination at 72°C for 30 s. The amplified PCR products were visualized under UV light after agarose gel (2%) electrophoresis.
164, 101, 34 and 8 bp, while the polymorphic A allele was confirmed by the presence of three fragments on the gel (164, 135 and 8 bp).
Measurement of oxytocin concentration
Plasma oxytocin concentration was measured by ELISA (ADI-900-153A, Enzo Life Sciences, Lausanne, Swit-zerland) according to the manufacturer’s instructions. Samples were diluted 1:8 and assayed without extrac-tion. Oxytocin concentration was expressed as pg/ mL. The minimum detectable dose (MDD) was 15.6 pg/ml. Intra- and interassay coefficients were <10%. Empathy Quotient (EQ) questionnaire
To measure empathy, each participant filled out the Serbian version of the self-report questionnaire, Empa-thy Quotient (EQ) [12]. The EQ questionnaire is based on a definition of empathy that includes all empathy components (cognition and affect). Participants rated each item from 1 (strongly agree) to 4 (strongly dis-agree). The results ranged from 0 to 80, with a higher score indicating a higher level of empathy.
Reading the Mind in the Eyes Test (RMET)
The RMET test is a behavioral measure of empathic accuracy which assesses individual differences in the ability to infer the affective mental states of strangers [13]. Participants were shown 36 black-and-white photographs where only the eye region of the face of different individuals was shown. The individual in each photograph displays a particular emotional or cognitive state. Each photo is paired with four affective-state adjectives as response options (e.g., “terrified”, “upset”, “arrogant” and “annoyed”). Participants select the adjective that according to their judgment best describes what the individual in the photo is feeling or thinking. The result is expressed as a total score, where the minimum score is 0 and the maximum score is 36. Statistical analysis
The frequency of alleles and genotypes in the males and females was analyzed and compared using the χ2
test or Fisher’s exact test; possible deviation from the
expected values of the Hardy-Weinberg equilibrium test for both studied polymorphisms was determined. The plasma oxytocin level, EQ score and RMET score were expressed as the mean (M)±standard deviation (SD). There was normality-assumption for all studied parameters. Statistically significant differences in values between males and females, as well as between different genotypes or diplotypes, were determined by the t-test for two independent samples or by analysis of variance (ANOVA). Pearson’s bivariate correlation analysis was performed to examine associations between plasma oxytocin levels and EQ. P<0.05 value was considered statistically significant. Statistical analysis was con-ducted using the SPSS software package version 20.0 (SPSS Inc., Chicago, IL, USA).
RESULTS
Genotype and allele frequencies of the rs53576
OXTR gene polymorphism
Genotype frequencies of the OXTR rs53576 and rs2254298 gene polymorphisms did not deviate from the normal distribution of the Hardy-Weinberg equi-librium (p>0.05). The results shown in Table 1 indicate that the rs53576 GG (wild-type) genotype was present in 37 (50%) subjects, while only 4 (5.4%) subjects had the AA genotype. Thirty-three (44.6%) subjects were heterozygous. The genotype frequency distributions of the rs53576 SNP in males were not significantly differ-ent from those in females (χ2=1.985, df=2, p=0.371). The difference was not detected in the frequency of the A allele in males compared to females (χ2=1.470, df=1, p=0.225; Fisher’s exact test p=0.267).
Table 1. Genotype and allele frequencies of the rs53576 OXTR
gene polymorphism.
Genotype
(rs53576) n=74All* Male*n=32 Female*n=42
Male vs Female p value
GG 37 (50.0%) 13 (40.6%) 24 (57.1%)
0.371 GA 33 (44.6%) 17 (53.1%) 16 (38.1%)
AA 4 (5.4%) 2 (6.3%) 2 (4.8%)
Allele 2n=148All 2n=64Male Female2n=84 p value G 107 (72.3%) 43 (67.2%) 64 (76.2%) 0.225 A 41 (27.7%) 21 (32.8%) 20 (23.8%)
Genotype and allele frequencies of the rs2254298
OXTR gene polymorphism
The results of rs2254298 SNP testing showed that the GG (wild-type) genotype was present in 65 (87.8%) sub-jects, GA in 8 (10.8%) subsub-jects, and AA only in 1 subject (1.4%). Neither the distribution of genotypes nor the A allele frequency of the rs2254298 polymorphism in males showed a statistically significant difference compared to females (χ2=0.919, df=2, p=0.632; χ2=0.766, df=1, p=0.381;
Fisher’s Exact test p=0.515 respectively; Table 2).
Table 2. Genotype and allele frequencies of the rs2254298 OXTR
gene polymorphism.
Genotype
(rs2254298) n=74All* Male*n=32 Female*n=42 Male vs Female
p value
GG 65 (87.8%) 29 (90.6%) 36 (85.7%) 0.919 GA 8 (10.8%) 3 (9.4%) 5 (11.9%)
AA 1 (1.4%) 0 (0.0%) 1 (2.4%) Allele All
2n=148 2n=64Male Female2n=84 p value G 138 (93.2%) 61 (95.3%) 77 (91.7%) 0.515 A 10 (6.8%) 3 (4.7%) 7 (8.3%)
*Hardy-Weinberg equilibrium p>0.05
The influence of gender on the EQ score, RMET score and plasma oxytocin levels
The results for the EQ score, RMET score and plasma oxytocin levels did not show a statistically significant difference between the tested parameters in male and female participants (EQ, p=0.115; RMET, p=0.573; oxytocin, p=0.502).
The influence of the rs53576 and rs2254298
OXTR SNPs on the tested parameters
Bearing in mind that the rs53576 AA genotype was detected in only 4 subjects and the rs2254298 AA geno-type in only 1 subject, we compared the EQ scores, RMET scores and oxytocin levels between GG genotype carriers (non A allele carriers) and GA/AA genotype carriers (A allele carriers) for both examined polymor-phisms. Subjects carrying the rs53576 GG genotype had a significantly higher EQ score compared to carriers of the polymorphic A allele (t=3.234, df=72, p=0.002). Analysis of RMET scores and oxytocin levels revealed no significant differences between different genotypes
of the rs53576 SNP (p=0.753; p=0.937 respectively). The results of the study of the rs2254298 SNP effect on the tested parameters showed that there were no significant differences between GG genotype carriers and GA/AA genotype carriers in the EQ score (p=0.803), RMET score (p=0.581) and oxytocin levels (p=0.207, Table 3).
Table 3. The influence of rs53576 and rs2254298 OXTR gene
poly-morphisms on the EQ score, RMET score and plasma oxytocin levels.
M
eas
ur
e rs53576 Genotypes rs2254298
GG (n=37) M (SD)
GA/AA (n=37) M (SD)
GG (n=65) M (SD)
GA/AA (n=9) M (SD)
EQ (7.99) *46.86 (6.84)41.27 (8.08)44.15 (6.82)43.44
RMET (3.45)25.02 (3.15)24.78 (3.27)24.98 (3.50)24.33 Oxytocin
(pg/ml) (131.33)354.85 (122.19)352.51 (119.35)360.60 (166.35)303.73
*p=0.002 GG vs rs53576 GA/AA
The influence of the OXTR SNPs on the tested parameters by gender
Taking into account the gender of the participants, the results of the study of the rs53576 SNP influence on the tested parameters showed that females carrying the GG genotype had a higher EQ score than female GA/AA genotype carriers (t=3.101, df=40, p=0.004). There was no difference in EQ score, RMET score or oxytocin levels between female and male participants, whether GG genotype (p=0.120, p=0.670, p=0.304) or GA/AA genotype carriers were analyzed (p=0.995, p=0.768, p=0.914 respectively, Table 4).
Table 4. The influence of rs53576 OXTR gene polymorphism on
the EQ score, RMET score and plasma oxytocin levels by gender.
M
eas
ur
e Genotypes (rs53576 polymorphism)GG GA/AA
Male (n=13) M (SD)
Female (n=24) M (SD)
Male (n=19) M (SD)
Female (n=18) M (SD)
EQ (8.04) 44.07 (7.70) *48.37 (7.06)41.26 (6.81)41.27
RMET (3.14)24.69 (3.65)25.20 (2.89)24.63 (3.48)24.94 Oxytocin
(pg/ml) (142.15)324.26 (125.05)371.41 (133.85)354.66 (112.43)350.25
The influence of gender on the EQ score (p=0.153), RMET score (p=0.210) and oxytocin levels (0.747) was not significant in rs2254298 GG genotype carriers. A significant difference was not detected in the EQ score (p=0.484), RMET score (p=0.108) and oxyto-cin levels (p=0.301) for rs2254298 GA/AA genotype carries either.
The influence of diplotypes of OXTR SNPs on the tested parameters
As regards diplotypes, there were statistically signifi-cant differences in the EQ score (F=3.626, p=0.017, η2 =0.135), while the RMET score and plasma oxytocin
levels did not show significant differences between the groups (F=1.464, df=3, p=0.232; F=1.275, df=3, p=0.290 respectively). A post hoc analysis showed that GGGG diplotype carriers (non A allele carriers for both polymorphisms) had a higher EQ score than GAGG/AAGG diplotype carriers (non A allele car-riers for rs2254298 polymorphism, mean difference MD=5.996, p=0.012, Table 5).
Table 5. The influence of diplotypes on the EQ score, RMET score
and plasma oxytocin levels.
M
eas
ur
e Diplotypes
GGGG (n=31) M (SD)
GAGG/ AAGG (n=34) M (SD)
GGGA/ GGAA (n=6) M (SD)
GAGA (n=3) M (SD)
EQ* (8.27)47.29 (6.86)41.29 (6.50)44.66 (8.18)41.00
RMET (3.37)25.41 (3.18)24.58 (3.40)23.00 (2.00)27.00 Oxytocin
(pg/ml) (128.93)356.40 (111.75)364.42 (156.06)346.83 (181.88)217.53
*p = 0.017 between groups, p=0.012 GGGG vs GAGG/AAGG
Correlations between the tested parameters
Correlation analyses between the EQ scores and oxy-tocin levels did not reveal significant associations in either the total sample of participants (r=0.12, p=0.922) or within the groups of rs53576 GG genotype carriers (r=0.164, p=0.332), of female rs53576 GG genotype carriers (r=0.020, p=0.931), and of GGGG diplotype carriers of the examined polymorphisms (r=0.265, p=0.149).
DISCUSSION
Although two polymorphisms (rs53576 and rs2254298) of the OXTR gene are often studied, to the authors’ best knowledge and based on a literature search, this is the first study to examine the impact of the two polymor-phisms on both empathy measures and oxytocin levels. In the present study, subjects carrying the GG genotype of OXTR rs53576 showed a significantly higher EQ score than A allele carriers. By gender analysis, signifi-cance was reached only in females. No difference was detected in the RMET score, as a measure of cognitive empathy, between different genotypes. The results of a meta-analysis of 24 studies [14] showed association between rs53576 GG genotype carriers and various forms of prosocial behavior. However, the results of the studies examining the effect of these SNPs on empathy are contradictory. Some studies did not find the effect of rs53576 SNP on empathy [10,15] when using self-rated Interpersonal Reactivity Index (IRI) scores. The results of other studies showed that subjects with the GG genotype exhibited higher trait empathy [9,16], the emotional component of empathy [4,16,17] and cogni-tive empathy [9], than carriers of the A allele; however, cognitive empathy was not found to be higher in wild-type carriers in the study of Uzefovsky et al. [4], with which our findings are in agreement. Our study did not show a difference in EQ and RMET scores between GG genotype carriers and A allele carriers of the OXTR
rs2254298 SNP. Conversely, the results of two studies [10,15] showed higher cognitive empathy in rs2254298 GG genotype carriers compared to A allele carriers. Differences in results could stem from the different questionnaires and tests used, as the abovementioned studies used the IRI, while we used the EQ question-naire and RMET. Our results were not consistent with the results of the study in which the RMET score was used [9]. It should be kept in mind that the distribution of polymorphisms varies depending on the sample size, genetic heterogeneity and ethnicity. In the study car-ried out by Rodrigues et al. [9], subjects had different ethnic origins, while our study included only Caucasian subjects. On the other hand, as an epigenetic factor, the cultural factor in different ethnic groups could affect behavior and cognitive empathy.
show statistically significant differences compared to the values detected in the carriers of GA/AA genotypes of both studied SNPs. The results of our study showed that there was no correlation between the EQ score and plasma oxytocin levels in any of the examined groups. In the study by Perry et al. [18], where subjects were administered intranasal oxytocin, in people with an EQ over 40 (highly empathic) this application stimulated the establishment of close interpersonal relationships, while the effect of oxytocin on people with low empathy was absent. The study by Hurlemann et al. [19] showed that the administration of oxytocin enhances emotional but not cognitive empathy. It was also shown that in autistic individuals carrying the G allele of OXTR
rs53576 and OXTR rs2254298 polymorphisms, the application of oxytocin was more effective compared to carriers of a polymorphic allele [20].
By examining the effect of the diplotypes of both of the tested polymorphisms on empathy and oxytocin levels, a significant difference in EQ scores between groups emerged, where GGGG diplotype carriers (non A allele carriers for both polymorphisms) had a significantly higher EQ score than GAGG/AAGG diplotype carriers (non A allele carriers for rs2254298 polymorphism), without a difference in plasma oxy-tocin levels.
Given the above, it is possible that the higher EQ score observed in this study in rs53576 GG genotype carriers (significantly in females), as well as in non A allele carriers for both polymorphisms (GGGG diplotype), was the result of hormone action that was not effected by oxytocin levels but by the number and function of the expressed receptors in the brain. Estrogen has been known to play an important role in OXTR expression [21]. We speculate that rs53576 GG genotype carriers, especially females, have higher expression of OXTR and a more efficient overall re-sponse to oxytocin binding in comparison to carriers of the A allele. However, this needs to be studied in more detail. Furthermore, OXTR rs53576 SNP could possibly influence the function of empathy-related brain areas, as neuroradiological methods used in the studies by Tost et al. [22] and Wang et al. [23] have shown that rs53576 G allele carriers have a larger amygdala and hypothalamus gray-matter volume and better func-tional association with the prefrontal cortex. Further studies of other variations in the OXTR gene in a larger
sample, as well as examination of the effects of SNP-SNP interactions on empathy, which we carried out in individuals for different SNPs [24,25], will clarify individual differences in the effects of this hormone.
The limitations of our study are related to the rela-tively small sample size. Plasma oxytocin levels show large daily variations, which we tried to eliminate by taking all the samples at the same time of day. Finally, even though our study was focused on oxytocin-related genetic effects on empathy, further investigation of this topic could include the effects of early environmental experiences as well.
In conclusion, higher empathy (EQ) in rs53576 GG genotype carriers and GGGG diplotype carriers in comparison with A allele carriers and GAGG/AAGG carriers, was not correlated with plasma oxytocin levels. These findings suggest that hormone effects might be due to an increased number and/or better function-ing of the OXTRs in the brain in non A allele carri-ers. However, this requires further and more detailed investigation in a larger sample of subjects.
Funding: This work was financially supported by the Ministry
of Education, Science and Technological Development of the Republic of Serbia (Grant No. III41018).
Author contributions: Jelena Bašić and Vuk Milošević designed
the experiment, performed the experiment, analyzed the data, contributed the reagents/materials/analysis tools, wrote the paper, prepared the tables and reviewed the drafts of the paper. Miloš Stanković designed the experiment, performed the experiment, analyzed the data, wrote the paper and reviewed the drafts of the paper. Tatjana Jevtović-Stoimenovdesigned the experiment, contributed the reagents/materials/analysis tools, performed the experiment and wrote the paper. Tatjana Cvetković contributed the reagents/materials/analysis tools, performed the experiment and wrote the paper. Milena Despotović performed the experiment, prepared the tables and wrote the paper. Dušica Pavlovićdesigned the experiment and reviewed the drafts of the paper.
Conflict of interest disclosure: The authors declare that there is
no conflict of interest.
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